The hybrid enzyme Sucrase-Isomaltase plays a crucial role in the digestion of oligosaccharides at the intestinal surface but the presumed intracellular site of its synthesis and the mechanisms of its transfer and insertion into the brush border surface membrane have not been defined. By use of an established solid-phase radioimmunoassay for rat sucrase-isomaltase, the various regions within the small intestinal villus and crypt cells such as the cytosol, endoplasmic reticulum and Golgi apparatus will be probed for the presence of a cross-reacting immunoprotein that may constitute the precursor of the fully formed membrane enzyme. The effect of specific inhibitors such as puromycin, Actinomycin D and Cytochalasin on the processing of the intracellular precursor will then be studied. The brush border membrane bound sucrase-isomaltase will be exposed to non-pentrating ligands such as 125I/lactoperoxidase, 3H-borohydride/periodate and 35S diazobenzene sulfonic acid to determine which domains of the enzyme are available at the luminal and cytoplasmic surface of the cell. After use of non-radioactive ligands for surface labeling, the intramembrane hydrophobic domains of the enzyme will be radiolabeled after solubilization with non-ionic detergents of urea. Depending on the results with the rat enzyme, similar experiments will be carried out for human intestine obtained from surgery or autopsy tissue. In this manner we expect to be able to obtain information about the site and sequence of synthesis, transfer and insertion of the sucrase-isomaltase molecule and the regulation of these processes.